1. Field of the Invention
This invention is in the field of genetic engineering. The invention is directed to a shortened phosphoglycerate kinase (PGK) promoter and the improvement it provides to the expression of polypeptides under its control. In addition, this invention is directed to the use of the shortened PGK promoter operably linked to a secretion signal that provides for improved secretion of the expressed proteins into the culture medium.
2. Background of the Invention
The glycolytic enzyme genes of Saccharomyces cerevisae encode some of the most abundant mRNA of protein species in the cell. The phosphoglycerate kinase (PGK) gene in the yeast genome has been extensively studied. Dobson et al., "Conservation of High Efficiency Promoter Sequences in Saccharomyces cerevisae," Nucleic Acids Research, 10: 2625-2637 (1982). The 5' control regions of the yeast glycolytic enzyme genes are particularly attractive for incorporation into yeast expression vectors as each gene encodes 1-5% of the total mRNA in protein. Further, the glycolytic genes are readily regulated by glucose. Thus, high level expression of a gene operably linked to the PGK promoter may be regulated by the simple control of the carbon source, glucose.
Researchers have identified approximately 1,500 nucleotides as the PGK 5' flanking region (the PGK promoter), derived from a 3 Kbp Hind III fragment. Using this full-length PGK promoter, the levels of heterologous protein produced under the control of the PGK promoter were not as high as expected for this promoter. Tuite et al., "Regulated High Efficiency Expression of Human Inteferon--Alpha in Saccharomyces cerevisae," EMBO Journal, 1: 603-608 (1982). Researchers have also reported that using the full length 5' DNA control regions of highly expressed yeast glycolytic genes, the protein levels produced were far less than that of the normal homologous gene product. In Chen et al., "Homologous v. Heterologous Gene Expression in the Yeast, Saccharomyces cerevisae," Nucleic Acids Research, 12: 8951-8970 (1984) the studies showed that the expression in yeast of heterologous genes adjacent to the PGK promoter on a high copy number plasmid vector were 15-50 times lower than the expression of the natural homologous gene on the same plasmid.
Applicants herein have discovered that using a PGK promoter with a shortened length of less than 500 base pairs improves the expression of polypeptides under its control. Thus, less time is required for transformants to appear, and there is an increase in polypeptide production.